The long-term objective of this proposal is to study the process of induced VanA-type vancomycin resistance in the opportunist Enterococcus faecium and to learn the sequence of events leading from exposure of cells to vancomycin to the activation of VanS, the environmental sensory of vancomycin. In the initial stages of work, the genes for VanS and VanR, the transcriptional regulatory element which is phosphorylated in response to VanS activation, will be expressed in Bacillus subtilis and Escherichia coli to obtain membrane-bound and over-expressed free protein for biochemical studies. In these preparations, antibodies will be tested for their ability to activate VanS, in vitro, free of background signals or spurious results which are obtained when antibiotics are used as inducers of resistance in vivo. The Specific Aims of this proposal are to develop in vitro systems to study molecular mechanisms of activation of the VanRS system. Specifically, we propose: (1) to develop a system with full-length VanS embedded in the cell membrane that has VanS-autophosphorylation, VanR-kinase, and phosphoVanR-phosphatase activities; and (2) to develop system with full-length VanS reconstituted into liposomes. These systems will be used to test mechanisms of VanS activation. The in vitro system with active full-length Van will allow us to address questions in three areas: (1) Can induction be reconstituted in vitro in a system in which the kinase and phosphatase activities are varied in response to inducing antibiotics? What attributes must an inducer possess? Does it have to be active as an antibiotic? Are there gratuitous inducers? (2) Does vancomycin interact directly with VanS? This possibility is suggested by reports that inactive glycopeptides can induce in a model fusion reporter system. (3) Can intermediates of cell wall peptidoglycan synthesis serve as inducers of VanS? This possibility is suggested by the observation that non-glycopeptide antibiotics induce resistance. Testing such intermediates will only be feasible in a cell-free system, where it is possible to have assurance that the effectors have access to their proposed targets.